20 research outputs found

    Basic mechanisms for control of appetite and feed intake in Atlantic salmon (Salmo salar)

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    Atlantisk laks (Salmo salar L.) er en nøkkelart i norsk sjømateksport, og representerer 70 – 80 Mrd NOK i verdi. Den store økningen i produksjon kombinert med overfôring har bidratt til negative effekter på det marine økosystemet. Av den grunn er effektiv utnyttelse av fiskefôret avgjørende for å sikre en bærekraftig produksjon med hensyn til miljø, fiskevelferd og produksjonskostnader (>50 % av produksjonskostnadene er til fôr). En økt forståelse av de grunnleggende biologiske mekanismene som styrer sult, metthetsfølelse, og kontroll av fôropptaket, vil gi mulighet til å forbedre dagens fôringsprotokoller og derav sikre økt fôrutnyttelse. Dette inkluderer kunnskap omkring de enkelte nevroendokrine faktorer som modulerer appetitt og fôringsatferd, samt å identifisere gode biomarkører som kan brukes til å vurdere appetitt og mulig vekst hos fisk. Hos virveldyr styres matinntaket av et samspill mellom sentrale og perifere signaler som inkluderer afferente nevroner og sirkulerende faktorer som hormoner i kombinasjon med signaler fra lukt- og syn og som bidrar til å stimulere fôrinntak i forhold til dyrets ernæringsstatus. Hypothalamus har en sentral rolle i å regulere appetitt og fôringsatferd. Hos pattedyr er det beskrevet et hypotalamisk nevronalt nettverk av store, distinkte cellepopulasjoner som uttrykker de oreksigene nevropeptidene (agouti-relatert protein (AgRP), nevropeptid Y (NPY)) og de anorexigene peptidene (proopiomelanocortin (POMC), kokain- og amfetaminregulert transkripsjon (CART)). Oreksigene peptider stimulerer appetitt og sult, mens anorexigene peptider stimulerer metthet, og inhiberer sultfølelse. Disse melanocortin-nevropeptidene virker på melanokortin-4-reseptoren (MC4R) i høyere ordens nevroner og som kontrollerer både matinntak og energiforbruk. Melanocortin systemet ser ut til å være relativt godt bevart blant virveldyr, inkludert teleoster. Hos atlantisk laks som har vært gjennom en laksefisk-spesifikk fjerde runde med hel genom duplikasjon (Ss 4WGD) vil de fleste av disse genene ha flere paraloge gener som kan ha ulike funksjoner for hver genvariant. Med dette som utgangspunkt har denne PhD-studien fokusert på følgende problemstillinger; 1. Å oppdatere dagens kunnskap bilde omkring tilstedeværelse av ulike sentrale appetittgener, 2. Å gjennomføre en distribusjonsanalyse av sentrale appetittgen paraloger i hjerneregionene bulbus olfactorius (OB), telencephalon (TEL), midthjernen (MB), cerebellum (CE), hypothalamus (HYP), saccus vasculosus (SV), hypofyse (PT) og hjernestamme (BS). Dette inkluderte også en vurdering av de ulike variantenes rolle i å kontrollere appetitten ved korttidsfaste (3 eller 4 dager) hos laks. 3. Å kartlegge hvordan appetittregulerende gener i mage-hypothalamus aksen påvirkes av langtidsfaste (4 og 6 uker) hos postsmolt av atlantisk laks med utgangspunkt i fordeling og uttrykk av de ulike paraloger i ulike avsnitt i hjernen hos laks. Innledningsvis ble det utført et ‘in silico’ søk for å hente sentrale appetittgener med paralogene og isoformer fra GenBank og Ensembl. I tillegg ble et fylogenetiske trær etablert. Deretter ble en standard protokoll for å dissekere fiskehjerne i 8 regioner utviklet for å studere fordelingen av de ulike genparaloger i fiskehjernen. Analyser for revers transkriptase- kvantitativ polymerasekjedereaksjon (RT-qPCR) ble utviklet m.h.p. sekvenssammenligningene og kvantifisering av mRNA-ekspresjon av appetittgenene. Parallelt ble identiteten til de nylig identifiserte sekvensene bekreftet ved å klone qPCR produkter. ‘In silico’ analyser bekreftet tilstedeværelsen av 2 agrp (agrp1 og 2 (også kjent som asip2)), 3 npy (npya1, a2 og b), 3 pomc (pomca1, a2 og b), 10 cart (cart1a, 1b1, 1b2, 2a, 2b1, 2b2, 3a1, 3a2, 3b og 4), 4 mc4r (mc4ra1, a2, b1 og b2) og 2 ghrl (ghrl1 og 2) genparaloger i genomdatabasen for atlantisk laks. Studien rapporterer den første identifiseringen og karakteriseringen av genparaloger for mc4r, npy, cart og ‘membrane-bound O-acyltransferase domain containing 4’ (mboat4) (Eng). Analyser av fordelingen av de analyserte genene i ulike regioner i hjernen viste et bredt distribusjonsmønster med varierende områder for alle de analyserte gener. HYP, TEL, OB, MB, PT og BS som viste et høyere mRNA-uttrykk blant de 8 hjerneregionene. agrp1, pomca1 og pomca2 var sterkt uttrykt i HYP, mens uttrykk av agrp2 var høyt i TEL og pomcb i PT. npya1 (mest rikelig blant npy) og npyb ble sterkt uttrykt i TEL og npya2 i HYP. Den mest uttrykte cart paralogen var cart2b, etterfulgt av cart3b, 3a, 1b, 2a, 4 og cart1a. cart2b var høyt uttrykt i OB og TEL, cart3b i BS og MB, cart3a i TEL, MB og BS, cart2a ble hovedsakelig uttrykt i MB og HYP, mens cart4 ble uttrykt i HYP og TEL. cart1a og 1b var hadde størst uttrykk i MB. agrp1, pomca1, pomca2, cart2a, 2b, 3a, 3b, 4 og npya2 viste et betydelig uttrykk i HYP i motsetning til de andre nevropeptidene. mc4rb1 var den høyest uttrykte mc4r-paralogen, og mc4ra1, a2 og b1 viste høyere uttrykk i HYP og TEL, mens mc4rb2 var høyt uttrykt i HYP. I forsøket med kortvarig (3 dagers) faste ble det registrert en signifikant oppregulering av transkripsjonsnivået i hypothalamus agrp1 i den fastede gruppen. Dessuten var mRNA uttrykket av agrp1 signifikant negativt korrelert med tørrvektsinnholdet i magen. Korrelasjonen mellom magefylling og agrp1 mRNA-uttrykket indikerer en sammenheng mellom magefylling og metthetssignaler. Dataene indikerer videre at hypothalamus agrp1 fungerer som et oreksigent signal hos laks. Etter 4 dagers Faste viste resultatene en signifikant reduksjon av npya1 mRNA uttrykket i OB, økt npya2 mRNA ekspresjon i midthjernen, og det ble også observert en trend med økt npya2 mRNA uttrykk i hypothalamus. Blant cart-paralogene, ble kun cart2b oppregulert etter fire dagers faste i OB, MB og HYP sammenlignet med gruppen som ble fôret. I forsøket hvor postsmolt laks ble fastet over en lengre periode (opp til 6 uker) viste resultatene at K faktoren avtok i Faste gruppen sammenlignet med Fôret gruppe. qPCR-analyse viste at mRNA uttrykket av agrp1 i hypothalamus ble signifikant oppregulert etter 4 og 6 ukers faste. Blant npy-paralogene npya1 og a2 var bare npya1-nivået signifikant oppregulert etter 4 ukers faste. Mens det blant cart paralogene ble registrert ett cart2a mRNA ekspresjonsnivå som var signifikant høyt etter 4 ukers faste. I tillegg økte cart2b-nivået statistisk i etter 4 og 6 ukers faste, mens cart3a og cart4 ikke responderte på faste. Paralogene pomca1, a2 og mc4ra2 økte signifikant etter 6 ukers faste og i magevev (etter 6 uker med faste) var det en tendens til at ghrelin1 (ghrl1) nivå ble redusert. Oppsummert m.h.p. de appetittregulerende genene som ble analysert så responderte agrp1, npya1, cart2a og 2b, pomca1 og a2, mc4ra2 og ghrl1 på matmangel både etter kortvarig og langvarig sult. Ved kortvarig faste spilte agrp1 i hypothalamus en tilsynelatende oreksigen rolle, og pomca2 viste en trend som tilsier en anoreksigen rolle. Under langtidsfaste, reagerte agrp1 og npya1 etter et oreksigent mønster, mens cart2a, cart2b, pomca1 og pomca2 ble oppregulert. Dette tyder på at disse nevropeptidene deltar aktivt i appetittreguleringen, dvs. faste kan indusere en nedstengning av sult og/eller motvirke sultsignaler (agrp1 og npya1) for å spare energi ved å redusere søkeatferd, eks. svømmeaktivitet under katabolske forhold der mat ikke er tilgjengelig. Alternativt kan disse nevropeptidene være en del av en sultindusert stressrespons. Basert på dataene kan en spekulere i at nedgangen i ghrl1 mRNA-uttrykk i magesekken under katabolske forhold forårsaket av matmangel, kan komme tilbake som sultsignal når mat igjen blir tilgjengelig. Samlet antyder dataene at agrp1 er en potensiell appetittbiomarkør, selv om den tidsmessige dynamikken til uttrykket i forhold til et måltid er komplisert og må undersøkes nærmere.Atlantic salmon (Salmo salar L.) is a key species in sea food exports of Norway and represents ca. 70 - 80 billion NOK worth annually. The extensive expansion of this fish industry combined with frequent overfeeding has contributed to potentially negative impacts on the ecosystem. As such, an efficient utilization of fish feed, with low feed conversion is vital to ensure sustainable production with regards to the environment, fish welfare, and production costs (>50% of production cost goes to feed). Thus, a clear understanding of the biological mechanisms that underly hunger and satiety, and thereby control of feed intake, would enable optimization of feeding protocols with a considerable positive impact to the salmon industry. The lack of understanding includes the contributions of key neuroendocrine players that modulate feeding and the identification of reliable biomarkers that can be used to assess appetite and potentially growth in fish. In vertebrates, food intake is controlled by the synergic actions of central and peripheral signals by afferent neurons and circulation in combination with olfactory and visual sensation, which stimulate ingestion in relation to the nutritional status of the animal. As a central control hub, the hypothalamus plays a pivotal role in regulating appetite and feeding. In mammals, hypothalamic neuronal network comprises major distinct cell populations that express the orexigenic neuropeptides (agouti-related protein (AgRP) and neuropeptide Y (NPY)) and the anorexigenic peptides (proopiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript (CART)). These melanocortin neuropeptides act on the melanocortin-4 receptor (MC4R) in higher order neurons that control both food intake and energy expenditure. The melanocortin system seems to be relatively well-conserved among vertebrates, including teleost species. However, in Atlantic salmon, the salmonid-specific fourth round whole-genome duplication (Ss 4WGD) led to the presence of several paralog genes which may have resulted in divergent functions for each of them. Therefore, this PhD study focused mainly on 1. Updating the key appetite genes repertoire information. 2. Spatial distribution analysis of key appetite gene paralogs in the brain regions bulbus olfactorius/olfactory bulb (OB), telencephalon (TEL), midbrain (MB), cerebellum (CE), hypothalamus (HYP), saccus vasculosus (SV), pituitary (PT) and brainstem (BS), including a first assessment of the appetite gene paralogs’ role in controlling appetite in short-term fasting (3 or 4 days) in Atlantic salmon. 3. Based on their spatial distribution and quantitative expression in different brain regions, the last focus was to explore the impact of long-term fasting (4 and 6 weeks) on appetite regulating genes in the stomach-hypothalamic axis in Atlantic salmon postsmolts. Initially, in silico analyses were performed to retrieve the sequences of the key appetite genes with paralogs and isoforms from GenBank and Ensembl and phylogenetic trees were predicted. Next, a standard protocol for dissecting fish brain into 8 regions was developed for the brain region distribution study of the key appetite genes. For quantification of mRNA expression of the appetite genes, assays for the retrieved sequences for reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) were developed and confirmed the identity of the novel sequences by cloning the qPCR product. In silico analyses have confirmed the presence of 2 agrp (agrp1 and 2 (also known as asip2)), 3 npy (npya1, a2 and b), 3 pomc (pomca1, a2 and b), 10 cart (cart1a, 1b1, 1b2, 2a, 2b1, 2b2, 3a1, 3a2, 3b and 4), 4 mc4r (mc4ra1, a2, b1 and b2) and 2 ghrl (ghrl1 and 2) gene paralogs in the Atlantic salmon genome database. This study reports the first identification and characterization of gene paralogs for mc4r, npy, cart and membrane-bound O-acyltransferase domain containing 4 (mboat4). Brain regional distribution analyses showed a wide distribution pattern with varying range for all genes analyzed. Among 8 brain regions HYP, TEL, OB, MB, PT and BS showed higher mRNA expressions. The agrp1, pomca1 and pomca2 were highly expressed in HYP while agrp2 was high in TEL and pomcb in PT. The npya1 (most abundant among npy) and b were highly expressed in TEL and npya2 in HYP. The cart2b was the most abundant cart paralog, followed by cart3b, 3a, 1b, 2a, 4 and cart1a. The cart2b was abundant in OB and TEL, cart3b in BS and MB, cart3a in TEL, MB, and BS and cart2a was in MB and HYP whilst cart4 in HYP and TEL. The cart1a and 1b were with higher expression in MB. The agrp1, pomca1, pomca2, cart2a, 2b, 3a, 3b, 4 and npya2 showed considerable expression in HYP in contrast to the other neuropeptides. The mc4rb1 was the most abundant mc4r paralog, and mc4ra1, a2 and b1 showed higher expression in HYP and TEL whilst mc4rb2 was highly expressed in HYP. In the short-term (3 days) fasting experiment a significant upregulation of hypothalamic agrp1 transcripts levels in the Fasted group was observed. Moreover, the mRNA abundance of agrp1 was significantly negatively correlated with the stomach dry weight content. The correlation between stomach fullness and agrp1 mRNA expression suggests a possible link between the stomach filling and satiety signals. The findings reported in this study indicates that hypothalamic agrp1 acts as an orexigenic signal in Atlantic salmon. Whereas 4 days of fasting resulted in a significant decrease in mRNA expression of npya1 in the olfactory bulb, increase of npya2 in the midbrain and a trend of increase of npya2 in the hypothalamus. Among cart paralogs only cart2b was upregulated after 4 days of fasting in OB, MB, and HYP compared to Fed group. In the long-term fasting experiment, the condition (K) factor of fish significantly decreased at both samplings for Fasted group compared to Fed group. In qPCR analysis, the hypothalamic relative mRNA expression of agrp1 showed highly significant upregulation at both 4 and 6 weeks of fasting. Among npy paralogs npya1 and a2, only npya1 level was significantly upregulated at 4 weeks of fasting. Whereas among cart paralogs cart2a mRNA expression level was significantly high only at 4 weeks fasting, cart2b level increased statistically in both 4 and 6 weeks of fasting, while cart3a and cart4 did not respond to fasting. The pomca1, a2 and mc4ra2 increased significantly at 6 weeks of fasting. Whereas in stomach, at 6 weeks of fasting ghrelin1 (ghrl1) declined. Conclusively, among the appetite regulating genes that was analyzed both in short-term and long-term fasting agrp1, npya1, cart2a & 2b, pomca1 & a2, mc4ra2 and ghrl1 responded to fasting. At short-term fasting hypothalamic agrp1 acted in an orexigenic role and pomca2 showed a trend of an anorexigenic role. Whereas in long-term fasting, the agrp1 and npya1 responded as orexigenic in action while cart2a, cart2b, pomca1 and pomca2 upregulation demonstrates that the neuropeptides might play either a vital role in appetite regulation i.e., fasting may be inducing shutting down of hunger and/or counteracting on hunger signals (agrp1 and npya1) to save energy from foraging search activity in catabolic conditions or play a fasting induced stress response. It is postulated that the decline in ghrl1 mRNA expression under catabolic conditions might return as hunger signal once the food is available in the vicinity. Taken together the data reported in this study suggest agrp1 as a potential appetite biomarker gene though the temporal dynamics of the expression in relation to a meal is complicated and need to be investigated further.Doktorgradsavhandlin

    Impact of long-term fasting on the stomach-hypothalamus appetite regulating genes in Atlantic salmon postsmolts

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    Atlantic salmon will experience periods of fasting during its lifecycle. In nature, prolonged fasting periods occur owing to seasonal fluctuations in available feeds, migration or in relation to reproduction. In a culture setting, salmon is fasted mainly as part of planned operational handling prior to vaccination, delousing, transfer etc., and where fasting may last up to nine days. The mechanisms regulating the appetite during long-term fasting may vary among fish species. Here, we studied the impact of long-term fasting on neuro-endocrine regulation of appetite through the stomach-hypothalamic axis in Atlantic salmon post smolts (1.2 kg, ∼46 cm), reared in two experimental conditions (Fed and Fasted; triplicated tanks), and sampled after 4 weeks and 6 weeks of fasting. Fasted fish showed lower condition factor and hepatosomatic index at both sampling points compared to Fed group. In qPCR analysis, hypothalamic relative mRNA expression of agouti-related protein 1 (agrp1) was upregulated in fasted group at both sampling points. Among neuropeptide Y (npy) paralogs, only npya1 at 4 weeks was upregulated by fasting. As for cocaine- and amphetamine-regulated transcripts (cart), cart2a was elevated at 4 weeks, and cart2b at both 4 and 6 weeks in fasted group, while cart3a and cart4 showed no response to fasting. The pro-opiomelanocortin (pomc) a1, a2 and melanocortin-4 receptor (mc4r) a2 increased only after 6 weeks of fasting, while mc4rb1 did not respond to fasting. In stomach, 6 weeks of fasting resulted in a decrease of ghrelin1 (ghrl1), while expression of mboat4 was unaffected. The elevated levels of hypothalamic agrp1 and npya1 in fasted group support orexigenic roles for these neuropeptides. In addition, upregulation of cart2a, cart2b, pomca1 and pomca2 indicate that these play vital roles in appetite regulation and that fasting may halt and/or counteract hunger signals (agrp1 and npya1) to save energy from foraging search activities during catabolic conditions. Another possibility is that these neuropeptides play a role in fasting-induced stress. Based on the drop in mRNA expression of ghrl under catabolic conditions, we hypothesize that Ghrl might return as hunger signal once feed becomes available. We also propose that agrp1 is a potential appetite biomarker gene under feed deprived conditions.publishedVersio

    Brain Distribution of 10 cart Transcripts and Their Response to 4 Days of Fasting in Atlantic Salmon (Salmo salar L.)

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    Cocaine- and amphetamine-regulated transcript (CART) has been known to be involved in feeding and energy balance in mammals, acting as an anorexigenic neuropeptide in hypothalamus. In Atlantic salmon, little is known about Cart brain localization and its function. In this study, in silico analysis revealed the existence of 10 cart paralogs, here named cart1a, 1b1, 1b2, 2a, 2b1, 2b2, 3a1, 3a2, 3b, and 4. The Atlantic salmon Cart sequences shared from 19 to 50% of identity with the human homolog and between 25 and 90% of sequence identity among paralogs, except for Cart4 which only shared 18–23% of identity. We further explored cart mRNA expressions in 8 brain regions (Olfactory Bulb-OB, Telencephalon-TEL, Midbrain-MB, Cerebellum-CE, Hypothalamus-HYP, Saccus vasculosus-SV, Pituitary-PT, and Brain Stem-BS) of Atlantic salmon smolt under 4 days of fasting and continuous fed conditions. The cart paralogs analyzed were widely distributed among the brain regions and OB, TEL, HYP, MB, and BS seemed to be the major sites of expression. The expression of cart1a and 1b showed quite similar pattern in MB, HYP, and BS. The expression of cart2a had the highest in MB followed by HYP and TEL. The cart3a transcript was widely distributed in rostrocaudal regions of brain except in OB and SV whereas cart3b was predominantly expressed in BS followed by MB. Expression of cart4 was high in HYP followed by TEL. With regards to effect of feeding status the Atlantic salmon cart2b, which is the most abundant among the paralogs, was upregulated after 4 days of fasting in OB, MB, and HYP compared to fed group. This may suggest an unexpected, but possible orexigenic role of cart2b in Atlantic salmon or a fasting induced stress effect. No other significant effect was observed. Collectively, the differential expressions of the cart paralogs in different brain regions suggest that they may have roles in regional integration of appetite signals and are possibly involved in regulating other brain functions in Atlantic salmon. The fact that salmon has 10 cart paralogs, while mammalians only one, opens interesting perspectives for comparative research on evolutionary adaptations of gene function in the control of appetite and energy homeostasis.publishedVersio

    Effects of short term fasting on mRNA expression of ghrelin and the peptide transporters PepT1 and 2 in Atlantic salmon (Salmo salar)

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    Food intake is a vital process that supplies necessary energy and essential nutrients to the body. Information regarding luminal composition in the gastrointestinal tract (GIT) collected through mechanical and nutrient sensing mechanisms are generally conveyed, in both mammals and fish, to the hypothalamic neurocircuits. In this context, ghrelin, the only known hormone with an orexigenic action, and the intestinal peptide transporters 1 and 2, involved in absorption of dietary di- and tripeptides, exert important and also integrated roles for the nutrient uptake. Together, both are potentially involved in signaling pathways that control food intake originating from different segments of the GIT. However, little is known about the role of different paralogs and their response to fasting. Therefore, after 3 weeks of acclimatization, 12 Atlantic salmon (Salmo salar) post-smolt were fasted for 4 days to explore the gastrointestinal response in comparison with fed control (n = 12). The analysis covered morphometric (weight, length, condition factor, and wet content/weight fish %), molecular (gene expression variations), and correlation analyses. Such short-term fasting is a common and recommended practice used prior to any handling in commercial culture of the species. There were no statistical differences in length and weight but a significant lower condition factor in the fasted group. Transcriptional analysis along the gastrointestinal segments revealed a tendency of downregulation for both paralogous genes slc15a1a and slc15a1b and with significant lowered levels in the pyloric ceca for slc15a1a and in the pyloric ceca and midgut for slc15a1b. No differences were found for slc15a2a and slc15a2b (except a higher expression of the fasted group in the anterior midgut), supporting different roles for slc15 paralogs. This represents the first report on the effects of fasting on slc15a2 expressed in GIT in teleosts. Transcriptional analysis of ghrelin splicing variants (ghrl-1 and ghrl-2) showed no difference between treatments. However, correlation analysis showed that the mRNA expression for all genes (restricted to segment with the highest levels) were affected by the residual luminal content. Overall, the results show minimal effects of 4 days of induced fasting in Atlantic salmon, suggesting that more time is needed to initiate a large GIT response.publishedVersio

    Protected sampling is preferable in bronchoscopic studies of the airway microbiome

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    The aim was to evaluate susceptibility of oropharyngeal contamination with various bronchoscopic sampling techniques. 67 patients with obstructive lung disease and 58 control subjects underwent bronchoscopy with small-volume lavage (SVL) through the working channel, protected bronchoalveolar lavage (PBAL) and bilateral protected specimen brush (PSB) sampling. Subjects also provided an oral wash (OW) sample, and negative control samples were gathered for each bronchoscopy procedure. DNA encoding bacterial 16S ribosomal RNA was sequenced and bioinformatically processed to cluster into operational taxonomic units (OTU), assign taxonomy and obtain measures of diversity. The proportion of Proteobacteria increased, whereas Firmicutes diminished in the order OW, SVL, PBAL, PSB (p<0.01). The alpha-diversity decreased in the same order (p<0.01). Also, beta-diversity varied by sampling method (p<0.01), and visualisation of principal coordinates analyses indicated that differences in diversity were smaller between OW and SVL and OW and PBAL samples than for OW and the PSB samples. The order of sampling (left versus right first) did not influence alpha- or beta-diversity for PSB samples. Studies of the airway microbiota need to address the potential for oropharyngeal contamination, and protected sampling might represent an acceptable measure to minimise this problem.publishedVersio

    Basic mechanisms for control of appetite and feed intake in Atlantic salmon (Salmo salar)

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    Atlantisk laks (Salmo salar L.) er en nøkkelart i norsk sjømateksport, og representerer 70 – 80 Mrd NOK i verdi. Den store økningen i produksjon kombinert med overfôring har bidratt til negative effekter på det marine økosystemet. Av den grunn er effektiv utnyttelse av fiskefôret avgjørende for å sikre en bærekraftig produksjon med hensyn til miljø, fiskevelferd og produksjonskostnader (>50 % av produksjonskostnadene er til fôr). En økt forståelse av de grunnleggende biologiske mekanismene som styrer sult, metthetsfølelse, og kontroll av fôropptaket, vil gi mulighet til å forbedre dagens fôringsprotokoller og derav sikre økt fôrutnyttelse. Dette inkluderer kunnskap omkring de enkelte nevroendokrine faktorer som modulerer appetitt og fôringsatferd, samt å identifisere gode biomarkører som kan brukes til å vurdere appetitt og mulig vekst hos fisk. Hos virveldyr styres matinntaket av et samspill mellom sentrale og perifere signaler som inkluderer afferente nevroner og sirkulerende faktorer som hormoner i kombinasjon med signaler fra lukt- og syn og som bidrar til å stimulere fôrinntak i forhold til dyrets ernæringsstatus. Hypothalamus har en sentral rolle i å regulere appetitt og fôringsatferd. Hos pattedyr er det beskrevet et hypotalamisk nevronalt nettverk av store, distinkte cellepopulasjoner som uttrykker de oreksigene nevropeptidene (agouti-relatert protein (AgRP), nevropeptid Y (NPY)) og de anorexigene peptidene (proopiomelanocortin (POMC), kokain- og amfetaminregulert transkripsjon (CART)). Oreksigene peptider stimulerer appetitt og sult, mens anorexigene peptider stimulerer metthet, og inhiberer sultfølelse. Disse melanocortin-nevropeptidene virker på melanokortin-4-reseptoren (MC4R) i høyere ordens nevroner og som kontrollerer både matinntak og energiforbruk. Melanocortin systemet ser ut til å være relativt godt bevart blant virveldyr, inkludert teleoster. Hos atlantisk laks som har vært gjennom en laksefisk-spesifikk fjerde runde med hel genom duplikasjon (Ss 4WGD) vil de fleste av disse genene ha flere paraloge gener som kan ha ulike funksjoner for hver genvariant. Med dette som utgangspunkt har denne PhD-studien fokusert på følgende problemstillinger; 1. Å oppdatere dagens kunnskap bilde omkring tilstedeværelse av ulike sentrale appetittgener, 2. Å gjennomføre en distribusjonsanalyse av sentrale appetittgen paraloger i hjerneregionene bulbus olfactorius (OB), telencephalon (TEL), midthjernen (MB), cerebellum (CE), hypothalamus (HYP), saccus vasculosus (SV), hypofyse (PT) og hjernestamme (BS). Dette inkluderte også en vurdering av de ulike variantenes rolle i å kontrollere appetitten ved korttidsfaste (3 eller 4 dager) hos laks. 3. Å kartlegge hvordan appetittregulerende gener i mage-hypothalamus aksen påvirkes av langtidsfaste (4 og 6 uker) hos postsmolt av atlantisk laks med utgangspunkt i fordeling og uttrykk av de ulike paraloger i ulike avsnitt i hjernen hos laks. Innledningsvis ble det utført et ‘in silico’ søk for å hente sentrale appetittgener med paralogene og isoformer fra GenBank og Ensembl. I tillegg ble et fylogenetiske trær etablert. Deretter ble en standard protokoll for å dissekere fiskehjerne i 8 regioner utviklet for å studere fordelingen av de ulike genparaloger i fiskehjernen. Analyser for revers transkriptase- kvantitativ polymerasekjedereaksjon (RT-qPCR) ble utviklet m.h.p. sekvenssammenligningene og kvantifisering av mRNA-ekspresjon av appetittgenene. Parallelt ble identiteten til de nylig identifiserte sekvensene bekreftet ved å klone qPCR produkter. ‘In silico’ analyser bekreftet tilstedeværelsen av 2 agrp (agrp1 og 2 (også kjent som asip2)), 3 npy (npya1, a2 og b), 3 pomc (pomca1, a2 og b), 10 cart (cart1a, 1b1, 1b2, 2a, 2b1, 2b2, 3a1, 3a2, 3b og 4), 4 mc4r (mc4ra1, a2, b1 og b2) og 2 ghrl (ghrl1 og 2) genparaloger i genomdatabasen for atlantisk laks. Studien rapporterer den første identifiseringen og karakteriseringen av genparaloger for mc4r, npy, cart og ‘membrane-bound O-acyltransferase domain containing 4’ (mboat4) (Eng). Analyser av fordelingen av de analyserte genene i ulike regioner i hjernen viste et bredt distribusjonsmønster med varierende områder for alle de analyserte gener. HYP, TEL, OB, MB, PT og BS som viste et høyere mRNA-uttrykk blant de 8 hjerneregionene. agrp1, pomca1 og pomca2 var sterkt uttrykt i HYP, mens uttrykk av agrp2 var høyt i TEL og pomcb i PT. npya1 (mest rikelig blant npy) og npyb ble sterkt uttrykt i TEL og npya2 i HYP. Den mest uttrykte cart paralogen var cart2b, etterfulgt av cart3b, 3a, 1b, 2a, 4 og cart1a. cart2b var høyt uttrykt i OB og TEL, cart3b i BS og MB, cart3a i TEL, MB og BS, cart2a ble hovedsakelig uttrykt i MB og HYP, mens cart4 ble uttrykt i HYP og TEL. cart1a og 1b var hadde størst uttrykk i MB. agrp1, pomca1, pomca2, cart2a, 2b, 3a, 3b, 4 og npya2 viste et betydelig uttrykk i HYP i motsetning til de andre nevropeptidene. mc4rb1 var den høyest uttrykte mc4r-paralogen, og mc4ra1, a2 og b1 viste høyere uttrykk i HYP og TEL, mens mc4rb2 var høyt uttrykt i HYP. I forsøket med kortvarig (3 dagers) faste ble det registrert en signifikant oppregulering av transkripsjonsnivået i hypothalamus agrp1 i den fastede gruppen. Dessuten var mRNA uttrykket av agrp1 signifikant negativt korrelert med tørrvektsinnholdet i magen. Korrelasjonen mellom magefylling og agrp1 mRNA-uttrykket indikerer en sammenheng mellom magefylling og metthetssignaler. Dataene indikerer videre at hypothalamus agrp1 fungerer som et oreksigent signal hos laks. Etter 4 dagers Faste viste resultatene en signifikant reduksjon av npya1 mRNA uttrykket i OB, økt npya2 mRNA ekspresjon i midthjernen, og det ble også observert en trend med økt npya2 mRNA uttrykk i hypothalamus. Blant cart-paralogene, ble kun cart2b oppregulert etter fire dagers faste i OB, MB og HYP sammenlignet med gruppen som ble fôret. I forsøket hvor postsmolt laks ble fastet over en lengre periode (opp til 6 uker) viste resultatene at K faktoren avtok i Faste gruppen sammenlignet med Fôret gruppe. qPCR-analyse viste at mRNA uttrykket av agrp1 i hypothalamus ble signifikant oppregulert etter 4 og 6 ukers faste. Blant npy-paralogene npya1 og a2 var bare npya1-nivået signifikant oppregulert etter 4 ukers faste. Mens det blant cart paralogene ble registrert ett cart2a mRNA ekspresjonsnivå som var signifikant høyt etter 4 ukers faste. I tillegg økte cart2b-nivået statistisk i etter 4 og 6 ukers faste, mens cart3a og cart4 ikke responderte på faste. Paralogene pomca1, a2 og mc4ra2 økte signifikant etter 6 ukers faste og i magevev (etter 6 uker med faste) var det en tendens til at ghrelin1 (ghrl1) nivå ble redusert. Oppsummert m.h.p. de appetittregulerende genene som ble analysert så responderte agrp1, npya1, cart2a og 2b, pomca1 og a2, mc4ra2 og ghrl1 på matmangel både etter kortvarig og langvarig sult. Ved kortvarig faste spilte agrp1 i hypothalamus en tilsynelatende oreksigen rolle, og pomca2 viste en trend som tilsier en anoreksigen rolle. Under langtidsfaste, reagerte agrp1 og npya1 etter et oreksigent mønster, mens cart2a, cart2b, pomca1 og pomca2 ble oppregulert. Dette tyder på at disse nevropeptidene deltar aktivt i appetittreguleringen, dvs. faste kan indusere en nedstengning av sult og/eller motvirke sultsignaler (agrp1 og npya1) for å spare energi ved å redusere søkeatferd, eks. svømmeaktivitet under katabolske forhold der mat ikke er tilgjengelig. Alternativt kan disse nevropeptidene være en del av en sultindusert stressrespons. Basert på dataene kan en spekulere i at nedgangen i ghrl1 mRNA-uttrykk i magesekken under katabolske forhold forårsaket av matmangel, kan komme tilbake som sultsignal når mat igjen blir tilgjengelig. Samlet antyder dataene at agrp1 er en potensiell appetittbiomarkør, selv om den tidsmessige dynamikken til uttrykket i forhold til et måltid er komplisert og må undersøkes nærmere

    Disintegration stability of extruded fish feed affects gastric functions in Atlantic salmon (Salmo salar)

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    In the present study, two salmon feeds with different disintegration stabilities were produced by impacting extruder viscous heat dissipation with the use of different lipid levels in the feed mix. The feeds were then dried and coated to similar final lipid levels (30-31%) and nutritional composition. Both feeds were produced as 4 and 6-mm pellets. Feeds extruded with 20% lipid had a lower disintegration stability compared with those with 8% lipid. The feeds were used in two feeding studies using Atlantic salmon (Experiments 1 and 2) and an in vitro gastric study (Experiment 3). In Experiment 1, salmon postsmolts (370 g starting weight) fed the 4-mm pellets showed the fastest gastrointestinal (GI) passage rate when fed feeds with a lower disintegration stability. This was due to a faster transport of content from the stomach to the gut at 30 min and 1.5 h after a meal, and faster passage through the distal gut 9–24 h after a meal. The 6-mm pellets were used in a 100-day feeding study (Experiment 2) in which salmon fed high and low disintegration stability feeds grew from 572 to 1542 and 1604 g, respectively. There was a higher total feed intake in salmon fed diets with lower disintegration stability, but only tendencies towards higher growth and final body weight compared with salmon fed diets with a higher disintegration stability. The in vitro gastric experiment showed that pellets with a lower disintegration stability required a larger supply of stomach acid to maintain a stable pH of 4.5, which also gave a lower content viscosity. The in vitro study also showed an increased release of water-soluble components from these pellets and hydrolysis of larger peptides increased the pool of intermediate-sized peptides (4–6 kDa) available for easy transportation, hydrolyzation, and absorption in the proximal intestine. Although lower pellet disintegration stability increases feed intake. The GI passage rate combined with an increased GI filling can affect the ability to hydrolyze and absorb nutrients. This may explain why an increased feed intake did not result in increased growth in Experiment 2, despite observing no significant differences in nutrient digestibility or feed conversion ratio. Our results highlight the importance of the quality of extruded fish feed.publishedVersio

    Drengenes_LaboratoryContamination_DRYAD_fastq

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    Fastq files generated by Illumina MiSeq sequencing of the bacterial 16S rRNA gene region V3V4 (164 samples, paired-end reads)

    Data from: Laboratory contamination in airway microbiome studies

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    Background: The low bacterial load in samples acquired from the lungs, have made studies on the airway microbiome vulnerable to contamination from bacterial DNA introduced during sampling and laboratory processing. We have examined the impact of laboratory contamination on samples collected from the lower airways by protected (through a sterile catheter) bronchoscopy and explored various in silico approaches to dealing with the contamination post-sequencing. Our analyses included quantitative PCR and targeted amplicon sequencing of the bacterial 16S rRNA gene. Results: The mean bacterial load varied by sample type for the 23 study subjects (oral wash>1st fraction of protected bronchoalveolar lavage>protected specimen brush>2nd fraction of protected bronchoalveolar lavage; p < 0.001). By comparison to a dilution series of know bacterial composition and load, an estimated 10-50% of the bacterial community profiles for lower airway samples could be traced back to contaminating bacterial DNA introduced from the laboratory. We determined the main source of laboratory contaminants to be the DNA extraction kit (FastDNA Spin Kit). The removal of contaminants identified using tools within the Decontam R package appeared to provide a balance between keeping and removing taxa found in both negative controls and study samples. Conclusions: The influence of laboratory contamination will vary across airway microbiome studies. By reporting estimates of contaminant levels and taking use of contaminant identification tools (e.g. the Decontam R package) based on statistical models that limit the subjectivity of the researcher, the accuracy of inter-study comparisons can be improved

    The Melanocortin System in Atlantic Salmon (Salmo salar L.) and Its Role in Appetite Control

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    The melanocortin system is a key neuroendocrine network involved in the control of food intake and energy homeostasis in vertebrates. Within the hypothalamus, the system comprises two main distinct neuronal cell populations that express the neuropeptides proopiomelanocortin (POMC; anorexigenic) or agouti-related protein (AGRP; orexigenic). Both bind to the melanocortin-4 receptor (MC4R) in higher order neurons that control both food intake and energy expenditure. This system is relatively well-conserved among vertebrates. However, in Atlantic salmon (Salmo salar L.), the salmonid-specific fourth round whole-genome duplication led to the presence of several paralog genes which might result in divergent functions of the duplicated genes. In the current study, we report the first comprehensive comparative identification and characterization of Mc4r and extend the knowledge of Pomc and Agrp in appetite control in Atlantic salmon. In silico analysis revealed multiple paralogs for mc4r (a1, a2, b1, and b2) in the Atlantic salmon genome and confirmed the paralogs previously described for pomc (a1, a2, and b) and agrp (1 and 2). All Mc4r paralogs are relatively well-conserved with the human homolog, sharing at least 63% amino acid sequence identity. We analyzed the mRNA expression of mc4r, pomc, and agrp genes in eight brain regions of Atlantic salmon post-smolt under two feeding states: normally fed and fasted for 4 days. The mc4ra2 and b1 mRNAs were predominantly and equally abundant in the hypothalamus and telencephalon, the mc4rb2 in the hypothalamus, and a1 in the telencephalon. All pomc genes were highly expressed in the pituitary, followed by the hypothalamus and saccus vasculosus. The agrp genes showed a completely different expression pattern from each other, with prevalent expression of the agrp1 in the hypothalamus and agrp2 in the telencephalon. Fasting did not induce any significant changes in the mRNA level of mc4r, agrp, or pomc paralogs in the hypothalamus or in other highly expressed regions between fed and fasted states. The identification and wide distribution of multiple paralogs of mc4r, pomc, and agrp in Atlantic salmon brain provide new insights and give rise to new questions of the melanocortin system in the appetite regulation in Atlantic salmon
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